The ability to mutate a cloned gene whose function in vivo is poorly understood is an important goal for mammalian developmental genetics. The newly emerging strategy for achieving this goal in mice depends not only on targeting a mutation to a given gene by homologous recombination between the endogenous gene and an introduced mutant copy, but also the ability to do so in cell type that retains its ability to form functional germ cells when placed into a host embryo. Embryo-derived stem (ES) cells are the cell type of choice because they are known to maintain their pluripotency during in vitro culture. Furthermore, these cells are capable of contributing to the germline of blastocyst-injection chimeras, from which mutant lines of mice can be established. Thus, gene targeting in ES cells offers exciting possibilities for determining the significance of genes that are expressed during development but whose functions are poorly understood. The long-term goal of the work outlined in this proposal is to interfere with expression of he gene that encodes the neural cell adhesion molecule (NCAM). NCAM is known to be a membrane-bound adhesion molecule that exists in multiple forms each of which are expressed in a temporal and cell-type specific pattern during development. A comprehensive and systematic series of experiments will be conducted to mutate the mouse NCAM gene by homologous recombination. Specific exons of the NCAM gene will be altered either by inserting a stop codon so that translation will be terminated, or by deleting specific exons from processed RNA by removing splice-acceptor sites. In either instance, the expression of specific forms of NCAM will be affected. Mice heterozygous for these mutations will be bred from founder chimeras produced by blastocyst-injection experiments. These mice will be crossed to produce homozygous embryos and the resulting phenotype examined.